Abstract
Several Lactobacillus ssp. are recognized as potential conjugated linoleic acid (CLA) producers. We have previously reported the ability of a range of Lactobacillus delbrueckii subsp. bulgaricus strains to produce CLA in fermented milk, being a potential candidate for the fermented dairy food chain. This study reports the draft genome sequence of L. bulgaricus strain LBP UFSC 2230, isolated from Italian Grana Padano cheese. Draft genome sequence originated in a total of 4,310,842 paired-end reads that were quality trimmed and assembled into 135 contigs with a total length of 604,745,873 bp, including 2086 protein coding genes and an average GC content of 49.7%. Draft genome sequence represents an important tool to identify the enzymes involved in this strain’s CLA metabolism. We identified a gene encoding an enzyme involved in biohydrogenation of linoleic acid pathway, oleate hydratase.
Keywords: Functional food; Bioprocess; Lactic acid bacteria; Lactobacillus ssp.; Genome sequencing; cis-9, trans-11 CLA
Introduction
Certain foods consist of a relevant number of functional microbial species. Among these, lactic acid bacteria (LAB) play an essential role in the development of starter cultures [1]. LAB are an important group of microorganisms used for a variety of food fermentations, and most of these LAB acquire the status of “Qualified Presumption of Safety” (QPS) or “Generally Recognized As Safe” (GRAS) by the European Food Safety Authority (EFSA) and by the US Food and Drug Administration (FDA), respectively. In particular, these LAB typically constitute a diverse group of Gram-positive, nonspore-forming, and nonmotile bacteria, producing lactic acid as major primary metabolite [2]. The use of these microorganisms and their enzymes for bioprocesses development has been extended to several bioactivities in the food industry, by enhancing nutrient bioavailability, providing flavor and texture [3], or converting unsaturated fatty acids into conjugated fatty acids, an example of which includes conjugated linoleic acid (CLA) [4] conversion.
CLA is a collective term used to designate a sequence of positional and geometric isomers of linoleic acid with conjugated double bonds. In particular, cis-9, trans-11 CLA is the major CLA isomer among Lactobacillus species and has been implicated as the most important isomer in terms of biological activity, such as anticarcinogenic [5], atherosclerotic [6], and body fat modulation [7]. A triple-component linoleic acid isomerase consisting of hydratase (CLA-HY), dehydrogenase/oxidoreductase (CLA-DH), and acetoacetate decarboxylase (CLA-DC), produced by Lactobacillus plantarum, was identified to convert linoleic acid into CLA [8].
Our previous analyses on CLA production in fermented milk demonstrated that Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230 is able to synthesize cis-9, trans-11 CLA [4]. In light of this, a genomic analysis was carried out to identify the genes responsible for CLA-HY, CLA-DH, and CLA-DC expression. In this study, we report phenotypic characterizations and the draft genome sequence of L. bulgaricus strain LBP UFSC 2230 isolated from Italian Grana Padano cheese. This strain has demonstrated a potent ability to produce CLA in dairy matrices and thus high biotechnological potential for use in CLA enrichment nourishments. The L. bulgaricus LBP UFSC 2230 strain presented as Gram-positive under optical microscopy, long rod-shaped and showed 2.7 h of generation time at 37 °C in the De Man, Rogosa, and Sharpe (MRS) broth without agitation. Colonies cultivated on MRS agar had a regular plain uncolored morphology with irregular borders.
Material and methods
Bacterial identification and phenotypic characterization
Preliminary microbial identification and phenotypic characterization regarding the metabolism of carbon sources were performed by using API 50CH (BioMérieux, France) (Table 1). The 16S rRNA gene sequence analysis was performed by Macrogen (Korea). Amplicons for the 16S rRNA sequences were generated using universal primers 1401R and 27F [9] on both strands. The consensus sequence of 1110 bp length was compared using the Basic Local Alignment Search Tool (BLAST) on the US National Center for Biotechnology Information (NCBI) Nucleotide Blast (BLASTn).
Table 1.
API 50CH fermentation profile of Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230
| Carbon source | Result | Carbon source | Result | Carbon source | Result |
|---|---|---|---|---|---|
| Glycerol | − | D-Mannitol | I | D-Raffinose | − |
| Erythritol | − | D-Sorbitol | − | Methyl-γD-Manopyranoside | − |
| D-Arabinose | − | Starch | I | Methyl-γD-Glucopyranoside | − |
| L-Arabinose | − | Glucogen | − | N-Acetylglucosamine | + |
| D-Ribose | − | Xylitol | − | Gentiobiose | − |
| D-Xylose | − | Amygdalin | − | D-Turanose | − |
| L-Xylose | − | Arbutin | + | D-Lyxose | − |
| D-Adonitol | − | Esculin | + | Methyl-βD-Xylopyranoside | − |
| Salicin | − | D-Tagatose | − | D-Fucose | − |
| D-Galactose | + | D-Cellobiose | − | L-Fucose | − |
| D-Glucose | + | D-Maltose | + | D-Arabitol | − |
| D-Fructose | + | D-Lactose | + | L-Arabitol | − |
| D-Mannose | + | D-Melibiose | − | Potassium gluconate | − |
| L-Sorbose | − | D-Saccharose | + | Potassium 2-Ketogluconate | − |
| L-Rhamnose | − | D-Trehalose | + | Potassium 5-Ketogluconate | − |
| Dulcitol | − | Inulin | − | Inositol | − |
| D-Melezitose | − |
(-) No fermentation up to 48 h of incubation; (+) Fermentation up to 48 h of incubation; (I) inconclusive.
Genome sequencing
Genome sequencing was carried out by WemSeq (Brazil). Genomic DNA was extracted using the UltraClean Tissue & Cells DNA Isolation Kit (Mo Bio, USA), and its integrity was ascertained visually in electrophoretic 1% agarose gel, while DNA purity and quantification were established by using Nanodrop (Thermo Scientific, USA) and Qubit (Invitrogen, USA), respectively. For library preparation, the Nextera XT kit (Illumina) was used according to the manufacturer’s instructions. Samples were sequenced on an Illumina MiSeq with a 500V2 kit 2 × 250 bp paired-end format. Total paired-end reads assembled from sequencing were trimmed for quality and de novo assembled using CLCBio Genomics Workbenchv8.5 (QIAGEN, Denmark) [10]. The average coverage of the genome was about 300×, and automatic annotation of resulting contigs was performed using RAST server [11].
Clustering unrooted tree
The evolutionary relationship was inferred by using the Maximum Likelihood method based on the Tamura-Nei model [12]. The tree with the highest log likelihood (− 17867.84) is shown. Initial tree (s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 13 nucleotide sequences represented by MLST concatemers for all 13 L. bulgaricus strains depicted in Table 2. Evolutionary analyses were conducted in MEGA X [13].
Table 2.
Complete genome sequences of Lactobacillus delbrueckii species available on GenBank
| GenBank BioProject no. | Microorganism | Strain | Genome size (Mb) | GC (%) | Genes | Proteins |
|---|---|---|---|---|---|---|
| PRJNA591475 | Lactobacillus delbrueckii | TS1-06 | 1.854 | 49.8 | 1928 | 1567 |
| PRJNA607413 | Lactobacillus delbrueckii subsp. bulgaricus | LJJ | 1.891 | 49.5 | 1948 | 1604 |
| PRJNA545627 | Lactobacillus delbrueckii subsp. bulgaricus | KLDS1.1011 | 1.887 | 49.8 | 1992 | 1634 |
| PRJNA304935 | Lactobacillus delbrueckii subsp. bulgaricus | MN-BM-F01 | 1.875 | 49.7 | 1933 | 1585 |
| PRJNA491249 | Lactobacillus delbrueckii subsp. bulgaricus | KLDS1.0207 | 1.869 | 49.8 | 1974 | 1607 |
| PRJNA354491 | Lactobacillus delbrueckii subsp. bulgaricus | DSM 20080 | 1.868 | 49.8 | 1942 | 1564 |
| PRJNA327771 | Lactobacillus delbrueckii subsp. bulgaricus | ND04 | 1.862 | 49.6 | 1932 | 1538 |
| PRJEB21527 | Lactobacillus delbrueckii subsp. bulgaricus | ACA-DC 87 | 1.856 | 49.8 | 1928 | 1579 |
| PRJNA331039 | Lactobacillus delbrueckii subsp. bulgaricus | L99 | 1.848 | 49.7 | 1944 | 1592 |
| PRJNA16120 | Lactobacillus delbrueckii subsp. bulgaricus | 2038 | 1.873 | 49.7 | 1941 | 1562 |
| PRJNA16871 | Lactobacillus delbrueckii subsp. bulgaricus | ATCC 11842 | 1.865 | 49.7 | 1940 | 1561 |
| PRJNA403 | Lactobacillus delbrueckii subsp. bulgaricus | ATCC BAA-365 | 1.857 | 49.7 | 1965 | 1579 |
| PRJNA49147 | Lactobacillus delbrueckii subsp. bulgaricus | ND02 | 2.132 | 49.59 | 2139 | 2011 |
| PRJNA615231 | Lactobacillus delbrueckii subsp. bulgaricus | LBP UFSC 2230 | 2.009 | 49.7 | 2165 | 2086 |
| PRJDB5979 | Lactobacillus delbrueckii subsp. delbrueckii | NBRC 3202 | 1.91 | 50.1 | 1930 | 1653 |
| PRJNA383854 | Lactobacillus delbrueckii subsp. delbrueckii | TUA4408L | 2.012 | 49.9 | 1999 | 1718 |
| PRJNA355324 | Lactobacillus delbrueckii subsp. delbrueckii | KCTC 13731 | 1.911 | 50 | 1905 | 1600 |
| PRJNA354490 | Lactobacillus delbrueckii subsp. delbrueckii | DSM 20074 | 1.954 | 49.6 | 1965 | 1577 |
| PRJNA355325 | Lactobacillus delbrueckii subsp. indicus | JCM 15610 | 2.022 | 49.37 | 2066 | 1939 |
| PRJNA355248 | Lactobacillus delbrueckii subsp. jakobsenii | DSM 26046 | 1.892 | 50.1 | 1941 | 1614 |
| PRJNA355327 | Lactobacillus delbrueckii subsp. lactis | KCCM 34717 | 2.263 | 49.1 | 2270 | 1905 |
| PRJNA398581 | Lactobacillus delbrueckii subsp. lactis | KCTC 3034 | 2.238 | 49 | 2240 | 1889 |
| PRJEB25625 | Lactobacillus delbrueckii subsp. lactis | 1 | 2.05 | 49.6 | 2072 | 1694 |
| PRJNA350761 | Lactobacillus delbrueckii subsp. lactis | KCTC 3035 | 1.973 | 50 | 1982 | 1697 |
| PRJNA454439 | Lactobacillus delbrueckii subsp. lactis | NWC_1_2 | 2.26 | 48.58 | 2297 | 2176 |
| PRJNA398701 | Lactobacillus delbrueckii subsp. lactis | DSM 20072 | 2.166 | 49 | 2141 | 1793 |
| PRJNA355244 | Lactobacillus delbrueckii subsp. sunkii | JCM 17838 | 2.004 | 50.1 | 1975 | 1726 |
| PRJNA356 | Lactobacillus plantarum | WCFS1 | 3.308 | 44.5 | 3042 | 3059 |
Nucleotide sequence accession numbers
Raw sequencing reads were deposited on the GenBank SRA database under accession no. SRR11741240. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JABSNS000000000. The version described in this paper is version JABSNS010000000.
Results and discussion
The phenotypic profile was not accurate since carbohydrate usage and fermentation was not fully compliant with the standards discriminated by the API test manufacturer, showing 36% Lactobacillus acidophilus and 27.5% Lactobacillus delbrueckii subsp. delbrueckii identity and T index values of 0.73 and 0.69, respectively. The phenotypic characterization of carbohydrate fermentation is shown in Table 1.
Since phenotypic identification of the strain was not successful, molecular identification was performed by 16S rRNA gene sequencing in which the consensus sequence returned a 98% query cover with 96.22% identity with L. bulgaricus strain LGM2 (GenBank accession no. AY675257.1).
Sequencing genome originated in a total of 4,310,842 paired-end reads that were quality trimmed and assembled into 135 contigs with a total length of 604,745,873 bp and an average GC content of 49.7%. The N50 of contig L. bulgaricus LBP UFSC 2230 genome is 39,590 bp, with the longest contig being 192,104 bp. The automatic annotation of the resulting contigs presented 2165 open reading frames (ORFs), of which 2,086 were protein-coding sequences and 79 were encoding RNAs. According to subsystem statistics of RAST shown in Fig. 1, 26% of features were categorized in subsystems while the majority of the genome could not be related to any subsystem by evidence. In this computation, there are 25 ORFs implicated in the category “Virulence, Disease and Defense”; however, all elements are present in the subcategory “Resistance to antibiotics and toxic compounds” of which only 5 are antibiotic-related.
Fig. 1.
Subsystem statistics generated by RAST automatic annotation
Subsystem computation by RAST covered only 26% of all present features including non-hypothetical and hypothetical genes in present subsystems. The majority of the features present in the L. bulgaricus LBP UFSC 2230 genome remains with uncertain functionality related to the categories above according to the automatic annotation pipeline. Nowadays (June 2020), there are 27 completed genomes available on the GenBank database from L. delbrueckii, and among them, 12 belong to the bulgaricus subspecies, which were isolated from yogurt or fermented milk, instead of cheese as did LBP UFSC 2230. There are low discrepancies among L. delbrueckii strains concerning GC content, but a number of genes annotated are widely variable between them, varying from 1905 up to 2297 genes (Table 2).
Song et al. [14] studied by using a multi-locus (8 genes) sequence typing (MLST) method, a collection with 298 L. bulgaricus strains obtained from naturally fermented products. The authors observed 106 Sequence Types (STs) grouped then into 6 Lineages (L) and 5 clonal complexes (CC). The genes encoding the following proteins were chosen for analysis: ATP-dependent protease ATP-binding subunit ClpX (clpX), chromosomal replication initiation protein (dnaA), CTP synthetase (pyrG), chaperon in GroEL (groEL), UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-L-lysine ligase (murE), phenylalanyl-tRNAsynthetase subunit alpha (pheS), recombinase A (recA), and DNA-directed RNA polymerase subunit beta (rpoB).
In order to verify similarity between available genomes of bulgaricus subspecies and LBP UFSC 2230 strain studied in the present work, a MLST analysis was also performed using the genes described by Song et al. [14]. The murE gene was excluded from the analysis because it was only present in half of the analyzed genomes. Unrooted phylogenetic trees were constructed from the concatenated MLST sequences (about 11.6 kb) using the maximum-likelihood method (Fig. 2). According to the generated tree, clusters were independent of the region of isolation of the strains (e.g., DSM20080 from Bulgaria and KLDS10207 from China instead of KLDS10207 and KLDS11011 both from China). Being the only strain obtained from cheese, LBP UFSC 2230 isolated in Italy has shown higher similarity to ND02, which was obtained from yogurt and isolated in the Inner Mongolia Autonomous Region.
Fig. 2.
Clustering MLST concatenated sequences of L. bulgaricus strains with completely sequenced genomes available in GenBank. Circles indicate the type of matrix from where strains were isolated from yogurt (yellow), traditional Chinese dairy product (blue), fermented milk (orange), starter culture (purple), and Grana Padano cheese (grey)
Regarding the biohydrogenation of LA, a possible metabolic pathway involving four enzymes, three of which are in an operon, was described [8]. Among the four genes described by the authors, only the hydratase is present in the sequenced genome of LBP UFSC 2230 strain. Comparing the genomic organization and especially the region related to the locus in where the hydratase gene is placed in LBP UFSC 2230 strain (Fig. 3), leads us to believe that the genomes of the species and subspecies of Lactobacillus tolerate wide rearrangements. Indeed, the same was suggested by Song and co-workers who reported that there has been significant evidence of frequent recombination across the whole subspecies [14].
Fig. 3.
Genome region comparison among LAB species. a Genome alignment and comparison among Lactobacillus plantarum WCSF1, Lactobacillus delbrueckii subsp. bulgaricus ND02, L. bulgaricus ND04, and L. bulgaricus LBP UFSC 2230 performed using Mauve [15]. Black arrow indicates position of CLA-DH, CLA-DC, and CLA-ER genes. Red arrows indicate position of CLA-HY. b Genome of L. plantarum WCSF1 indicating CLA-HY, CLA-DH, CLA-DC, and CLA-ER positions. Red arrow indicates both enzymes belonging to oleate hydratase superfamily (DH, dehydrogenase; HY, hydratase/dehydratase) while black arrows indicate other classes of enzymes (DC, isomerase; ER, desaturase) involved in linoleic acid biohydrogenation. c Oleate hydratase (red arrows) found in the L. bulgaricus LBP UFSC 2230 strain genome (first genome) compared to Lactobacillus acidophilus NCFM (two regions), Lactobacillus reuteri JCM 1112, and Lactobacillus gasseri ATCC 33323 (two regions) available on RAST database
Conclusion
Metabolic pathway that generates CLA in L. bulgaricus LBP UFSC 2230 remains unclear, once we only identified one gene encoding an enzyme involved in the biohydrogenation of linoleic acid. Further studies will be carried out in order to establish the role of oleate hydratase in CLA production in the strain. The use of LAB as a source of enzymes or as a heterologous host could have significant advantages in the food industry. Understanding these LAB and their enzymes allow for the generation of novel functional products, presenting a large opportunity for research and industry.
Repository GenBank Accession number: BioProject accession number PRJNA615231 (SRA accession number SRR11741240, WGS accession number JABSNS010000000)
Acknowledgements
We are grateful to Dr. Fabienne Ferreira (Department of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina, Brazil), Dr. Harsh Mathur (Teagasc Food Reasearch Centre, Ireland) for useful comments on this manuscript, and Iwona Kozak, MSc (APC Microbiome Ireland, Biosciences Institute, University College Cork, Ireland) for contributing with the raw data GenBank submission.
Funding
This research was supported by the Brazilian federal government agency Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), financial code 001.
Declarations
Conflict of interest
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Gabriela Christina Kuhl and Ricardo Ruiz Mazzon contributed equally to this work.
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